This patent application claims foreign priority benefits. Specifically, this patent application claims the benefit of the filing date under 35 U.S.C. 119 of Japanese Application No. 2000-305279, filed Oct. 4, 2000.
The present invention relates to a protein having an antithrombotic activity and a method for producing the same. The present invention also relates to DNA coding for the protein and a drug containing the protein as an active ingredient.
The number of patients with thromboses such as myocardial infarction and cerebral thrombosis, in particular, arterial thrombosis, is high in the world, and these are very important diseases to be treated. In an early stage of onset of arterial thrombosis, von Willebrand factor in blood binds to subendothelial tissues (collagen etc.) exposed due to impairment of vascular endothelial cells, and a membrane glycoprotein on platelets, glycoprotein Ib, binds to the von Willebrand factor. Thus, the platelets adhere to blood vessel walls and they are activated (J. P. Cean et al., J. Lab. Clin. Med., 87, pp.586-596, 1976; K. J. Clemetson et al., Thromb. Haemost., 78, pp.266-270, 1997). Therefore, it is an important target for antithrombotic drugs for treating or preventing thromboses to inhibit the binding of von Willebrand factor and glycoprotein Ib. However, there are few substances that have proven to exhibit antithrombotic property by inhibiting the binding of these proteins, and such drugs have not been used in clinical practice. It has been reported that a recombinant protein VCL that has a sequence of the 504th to 728th amino acid residues of the amino acid sequence of von Willebrand factor shows an antithrombotic action by inhibiting the binding of von Willebrand factor and glycoprotein Ib (K. Azzam et al., Thromb. Haemost., 73, pp.318-323, 1995). Further, it has also been reported that a monoclonal antibody AJvW-2 directed to human von Willebrand factor exhibits an antithrombotic activity by specifically binding to von Willebrand factor without showing hemorrhagic tendency (S. Kageyama et al., Br. J. Pharmacol., 122, pp.165-171, 1997). It has also been shown that a monoclonal antibody 6B4 directed to glycoprotein Ib has an antithrombotic action in animal models (N. Cauwenberghs et al., Atherioscler. Thromb. Vasc. Biol., 20, pp.1347-1353, 2000). Furthermore, the protein AS1051 originating from snake venom specifically binds to platelet glycoprotein Ib to similarly exhibit antithrombotic property without showing hemorrhagic tendency (N. Fukuchi et al., WO95/08573).
Meanwhile, the binding of von Willebrand factor and glycoprotein Ib is not observed under a usual condition, but is considered to occur only under a condition where shear stress is induced, such as a condition in a blood flow (T. T. Vincent et al., Blood, 65, pp.823-831, 1985). However, as a method for artificially observing the binding of these proteins, there are known addition of an antibiotic, ristocetin (M. A. Howard and B. G. Firkin, Thromb. Haemost., 26, pp.362-369, 1971) and addition of a protein originating from snake venom, botrocetin (M. S. Read et al., Proc. Natl. Acad. Sci. USA., 75, pp.4514-4518, 1978). That is, both of the substances are considered to cause a structural change of von Willebrand factor by binding to a specific site of von Willebrand factor, thereby causing the binding of von Willebrand factor and glycoprotein Ib, which does not occur under a usual condition.
As proteins originating from snake venom, there are known, in addition to the aforementioned AS1051 (derivative of the xcex1-chain of a protein originating from Crotalus horridus horridus snake venom, CHH-B) and its original protein, CHH-B, many glycoprotein Ib-binding proteins such as alboaggregin, echicetin, mamushigin, jararaca-GPIbBP and proteins originating from Cerastes cerastes. Many of these proteins have a heterodimeric structure, and the amino acid sequences of their subunits show a homology of not less than 30%. Furthermore, they are proteins in which all of subunits show an amino acid sequence homology of not less than 30% to the CHH-B xcex1-chain (R. K. Andrews et al., Biochemistry, 35, pp.12629-12639, 1996; Y. Fujimura et al., Thromb. Haemost., 76, pp.633-639, 1996).
While such glycoprotein Ib-binding proteins originating from snake venom that inhibit the binding of glycoprotein Ib and von Willebrand factor and monoclonal antibodies directed to von Willebrand factor or glycoprotein Ib are known to exhibit an antithrombotic action as described above, some of proteins originating from snake venom that bind to a platelet membrane glycoprotein, glycoprotein IIb/IIIa, disintegrins (T. Matsui et al., Biochem. Biophys. Acta, 1477, pp.146-56, 2000) and monoclonal antibodies directed to glycoprotein IIb/IIIa (A. M. Lincoff et al., J. Am. Coll. Cardiol., 35, pp.1103-1115, 2000) have also been shown to exhibit an antithrombotic activity in animal experiments or clinical practice. For example, a peptide prepared from a disintegrin sequence, Eptifibatide (integrilin), has been shown to have clinical efficacy as an antithrombotic drug. Further, a chimerized monoclonal antibody directed to glycoprotein IIb/IIIa, Abciximab (ReoPro), is also widely used as an antithrombotic drug in clinical practice and its strong antithrombotic action and its therapeutic action for acute coronary syndromes have been reported (M. Madan et al., Circulation, 98, pp.2629-2635, 1998).
In addition to the above proteinaceous substances, low molecular weight organic compounds that bind to a platelet membrane glycoproteins and inhibit their function are known with respect to glycoprotein Ib (N. Fukuchi et al., WO99/54360; W. Mederski et al., WO00/32577; H. Matsuno et al., Circulation, 96, pp.1299-1304, 1997) and glycoprotein IIb/IIIa (E. J. Topol et al., Lancet, 353, pp.227-231, 1999). Among these, some of glycoprotein IIb/IIIa antagonists are clinically used, but they have not been shown to have efficacy as high as that of Abciximab (ReoPro) (E. J. Topol et al., Lancet, 353, pp.227-231, 1999; M. Madan et al., Circulation, 98, pp.2629-2635, 1998).
As described above, proteins that bind to platelet membrane proteins involved in thrombogenesis such as glycoprotein Ib and glycoprotein IIb/IIIa and inhibit their functions in thrombogenesis are useful as antithrombotic drugs, and many exogenous proteins have been developed as antithrombotic drugs. Among these, a chimerized monoclonal antibody directed to glycoprotein IIb/IIIa, Abciximab (ReoPro), shows high clinical efficacy. However, the following some conditions are still required to use proteinaceous substances, in particular, exogenous proteins as clinically usable drugs.
(1) High Binding Activity to Target
In the case of Abciximab (ReoPro), a high binding activity to platelets (glycoprotein IIb/IIIa) can be mentioned as one of the reasons for its high efficacy (R. M. Scarborough et al., Circulation, 100, pp.437-444, 1999). That is, it is considered that the administered Abciximab (ReoPro) firmly binds to platelets and as a result, it exists in blood together with platelets for a long period, thereby showing drug efficacy for a long period.
(2) Long Half-life/High Drug Efficacy Retention in Blood
For administration of a proteinaceous drug, in particular, a drug that is not originally an endogeneous substance existing in the organisms, repetitive administration is generally difficult and a single administration is usually performed. Therefore, drug efficacy must be maintained for a certain long period and long half-life and/or high drug efficacy retention in blood is required.
(3) Low Antigenicity
Even when a single administration is performed, low antigenicity is required so that an excessive antigen-antibody reaction should not occur.
(4) Useful Actions in Addition to Main Action
There have been reported that Abciximab (ReoPro) actually has a binding action directed to other proteins such as xcex1vxcex23 integrin and Mac-1 in addition to an inhibitory action directed to glycoprotein IIb/IIIa (B. S. Coller, Thromb. Haemost., 82, pp.326-336, 1999). It is considered that this secondary action is one of the reasons for high clinical efficacy. That is, clinical efficacy of a drug may be increased by acting on several targets other than a single target.
It has been reported that a drug for inhibiting the binding of glycoprotein Ib and von Willebrand factor has a low risk of hemorrhage compared with a drug for inhibiting the function of glycoprotein IIb/IIIa (S. Kageyama et al., Br. J. Pharmacol., 122, pp.165-171, 1997), and therefore it can be a useful antithrombotic drug. Among the aforementioned proteins that inhibit the binding of glycoprotein Ib and von Willebrand factor, monoclonal antibodies generally have a high binding activity (affinity) to a target and can satisfy the above requirements (2) and (3) if they are modified into a chimera antibody or a humanized antibody. On the other hand, it is considered that proteins other than the monoclonal antibodies, for example, a glycoprotein Ib-binding protein originating from snake venom have a low binding activity (affinity) to their targets. For example, when the anti-platelet activity disclosed for a protein derivative originating from snake venom, AS1051 (N. Fukuchi et al., WO95/08573), is compared with that of a monoclonal antibody, AJvW-2 (S. Kageyama et al., Br. J. Pharmacol., 122, pp.165-171, 1997), the binding activity (affinity) of AS1051 on a molar concentration basis is calculated to be about {fraction (1/10)} based on the fact that the efficacy is shown at almost the same concentration (weight concentration), and the molecular weight of AS1051 is about 15,000 Da and that of the monoclonal antibody about 150,000 Da.
Further, the present inventors found that, as shown in the examples described later, repetitive administration of AS1051 produces antibodies for AS1051 as an antigen and subsequent administration thereof caused platelet decrease that was considered to be attributable to the antibody generation.
That is, in order to clinically use glycoprotein Ib-binding proteins originating from snake venom such as AS1051 as antithrombotic drugs, they must further be improved for the aforementioned requirements (1) to (3).
The inventors of the present invention successfully elucidated a crystal structure of a glycoprotein Ib-binding protein originating from snake venom, AS1051, by preparing crystals of a specific mutant AS1051 and analyzing them by X-ray diffraction analysis, and thus identified a structure unique to AS1051. Moreover, they successfully improved glycoprotein Ib-binding proteins such as AS1051 by modifications based on the structure. That is, they found a method for improving a protein so that the protein should satisfy the aforementioned four kinds of properties, which were considered to be required to use an exogenous protein as a clinically applicable drug, i.e., (1) a high binding activity to target, (2) long half-life/drug efficacy retention in blood, (3) low antigenicity and (4) useful actions, in addition to its main action, as well as such an improved protein. Thus, they accomplished the present invention.
The present invention provides a method for producing a protein having an antithrombotic activity, which comprises replacing, in a protein that has an amino acid sequence having a homology of not less than 30% to the amino acid sequence of SEQ ID NO: 1 and forms a higher order structure composed of a first xcex2 strand (xcex21), a first xcex1 helix (xcex11), a second xcex1 helix (xcex12), a second xcex2 strand (xcex22), a loop, a third xcex2 strand (xcex23), a fourth xcex2 strand (xcex24) and a fifth xcex2 strand (xcex25) in this order from the amino terminus, at least one amino acid residue in a region from xcex12 to xcex22 and/or a region from xcex23 to xcex24 so that electric charge of the amino acid residue is changed towards positive direction (hereafter also referred to as the xe2x80x9cproduction method of the present inventionxe2x80x9d).
In the production method of the present invention, electric charge is preferably changed towards positive direction by replacing at least one acidic amino acid residue in the region from xcex12 to xcex22 and/or the region from xcex23 to xcex24 with a neutral amino acid residue.
In the production method of the present invention, the protein preferably originates from Crotalus horridus horridus. 
Further, it is preferred that the region from xcex12 to xcex22 in the protein corresponds to the sequence of the amino acid numbers 47 to 72 in the amino acid sequence of SEQ ID NO: 1 and the region from xcex23 to xcex24 corresponds to the sequence of the amino acid numbers 94 to 111 in the amino acid sequence of SEQ ID NO: 1. In this embodiment, it is preferred that at least one acidic amino acid residue of which a carbon atom exists within 10 xc3x85 from the xcex1 carbon atom of the arginine residue of the amino acid number 103 in the amino acid sequence of SEQ ID NO: 1 is replaced with a neutral amino acid residue. Further, the acidic amino acid residue preferably is at least one residue selected from the aspartic acid residue of the amino acid number 54, the aspartic acid residue of the amino acid number 101 and the glutamic acid residue of the amino acid number 106 in the amino acid sequence of SEQ ID NO: 1.
The production method of the present invention may further comprise deleting a region containing the loop structure existing between xcex22 and xcex23 in such a manner that the higher order structures of xcex22 and xcex23 are maintained, or replacing the region with one or more amino acid residue(s) in a number required to maintain the higher order structures of xcex22 and xcex23, said amino acid residue(s) being selected from the group consisting of a glycine residue, an alanine residue, a serine residue and a cysteine residue. Preferably, the region containing the loop structure existing between xcex22 and xcex23 is replaced with an amino acid sequence composed of four glycine residues.
The production method of the present invention preferably further comprises bonding a polyoxyalkylpolyol group to the protein. Preferably, the protein contains a cysteine residue corresponding to a cysteine residue of the amino acid number 81 in the amino acid sequence of SEQ ID NO: 1, and the polyoxyalkylpolyol group is bonded to this cysteine residue. The polyoxyalkylpolyol group is preferably a polyethylene glycol group.
The present invention also provides a protein having an antithrombotic activity, which has an amino acid sequence showing a homology of not less than 30% to the amino acid sequence of SEQ ID NO: 1 and forms a higher order structure composed of a first xcex2 strand (xcex21), a first xcex1 helix (xcex11), a second xcex1 helix (xcex12), a second xcex2 strand (xcex22), a loop, a third xcex2 strand (xcex23), a fourth xcex2 strand (xcex24) and a fifth xcex2 strand (xcex25) in this order from the amino terminus, and wherein at least one amino acid residue in a region from xcex12 to xcex22 and/or a region from xcex23 to xcex24 is replaced so that electric charge of the amino acid residue in the regions is changed towards positive direction, said protein being the following (a) or (b) (hereafter also referred to as the xe2x80x9cprotein of the present inventionxe2x80x9d):
(a) a protein, in which the region from xcex12 to xcex22 has the sequence of the amino acid numbers 47 to 72 in the amino acid sequence of SEQ ID NO: 1 and the region from xcex23 to xcex24 has the sequence of the amino acid numbers 94 to 111 in the amino acid sequence of SEQ ID NO: 1;
(b) the protein according to (a), in which substitution, insertion or deletion of one or several amino acid residues is included in the region from xcex12 to xcex22 having the sequence of the amino acid numbers 47 to 72 in the amino acid sequence of SEQ ID NO: 1 and/or the region from xcex23 to xcex24 having the sequence of the amino acid numbers 94 to 111 in the amino acid sequence of SEQ ID NO: 1.
The protein of the present invention preferably comprises an amino acid sequence of the following (A) or (B):
(A) the amino acid sequence of the amino acid numbers 47 to 111 in the amino acid sequence of SEQ ID NO: 1;
(B) the amino acid sequence according to (A), in which the cysteine residue of the amino acid number 81 in the amino acid sequence of SEQ ID NO: 1 is replaced with an alanine residue.
The protein of the present invention may have the amino acid sequence in which a region containing the loop structure existing between xcex22 and xcex23 is deleted in such a manner that the higher order structures of xcex22 and xcex23 are maintained, or the region is replaced with one or more amino acid residue(s) in a number required to maintain the higher order structures of xcex22 and xcex23, said amino acid residue(s) being selected from the group consisting of a glycine residue, an alanine residue, a serine residue and a cysteine residue. The region preferably has the sequence in which the region containing the loop structure existing between xcex22 and xcex23 is replaced with an amino acid sequence composed of four glycine residues.
The protein of the present invention preferably has a sequence in which at least one acidic amino acid residue of which a carbon atom exists within 10 xc3x85 from the xcex1 carbon atom of the arginine residue of the amino acid number 103 in the amino acid sequence of SEQ ID NO: 1 is replaced with a neutral amino acid residue. In this embodiment, it is preferred that the acidic amino acid residue to be replaced is composed of at least one residue selected from the aspartic acid residue of the amino acid number 54, the aspartic acid of the amino acid number 101 and the glutamic acid residue of the amino acid number 106 in the amino acid sequence of SEQ ID NO: 1.
Preferably, the protein of the present invention is bonded to a polyoxyalkylpolyol group. The protein of this embodiment preferably contains a cysteine residue corresponding to the cysteine residue of the amino acid number 81 in the amino acid sequence of SEQ ID NO: 1, and the polyoxyalkylpolyol group is bonded to this cysteine residue. The polyoxyalkylpolyol group is preferably a polyethylene glycol group.
The present invention also provides a DNA coding for the protein of the present invention (hereafter also referred to as the xe2x80x9cDNA of the present inventionxe2x80x9d), as well as a method for producing the protein of the present invention, which comprises steps of culturing a host microorganism transformed with the DNA of the present invention and collecting a protein encoded by the DNA from a culture and a method for producing the protein of the present invention, which comprises steps of culturing a host microorganism transformed with the DNA of the present invention, collecting a protein encoded by the DNA from a culture and bonding a polyoxyalkylpolyol group to the collected protein.
The present invention further provides a drug containing the protein of the present invention as an active ingredient. Also, the present invention provides a pharmaceutical composition comprising the protein of the present invention and a pharmaceutically acceptable carrier, and a use of the protein of the present invention for the manufacture of a medicament.
According to the present invention, a glycoprotein Ib-binding protein originating from snake venom can be improved to obtain a protein having (1) higher activity, (2) higher drug efficacy retention, (3) lower antigenicity, (4) thrombin-induced aggregation inhibitory action in addition to its main action, i.e., an inhibitory activity for binding of glycoprotein Ib and von Willebrand factor, and so forth, and the improved protein can be utilized as a more effective antithrombotic drug.